This invention is directed to methods for detecting analytes in biological fluids, typically using a combination of capillary electrophoresis and an enzymatic reaction.
For many purposes, it is important to detect analytes in biological fluids such as urine, plasma, lymphatic fluid, or cerebrospinal fluid. In many cases, particular analytes are markers of disease conditions, and the disease condition is detected either by the presence of an otherwise abnormal analyte or the presence of an abnormal concentration of an otherwise normally occurring analyte. For example, the amino acid phenylalanine is present in abnormally high concentrations in the serum of individuals with the genetic disorder phenylketonuria, which can cause severe mental retardation in untreated individuals. Thus, accurate diagnosis of this genetic disorder is essential. Other aminoacidurias are also inheritable genetic disorders and can result in a wide variety of serious consequences. These disorders are treated by special diets, and it is therefore essential that the presence of the disease be diagnosed accurately.
Another example of an analyte that is frequently assayed for clinical purposes is uric acid. Uric acid is a breakdown product of purines, and is a normal constituent of serum and urine. However, higher than normal concentrations of uric acid in serum are characteristic of gout, which can cause arthritis and renal disease. Other syndromes, such as Lesch-Nyhan Syndrome, are also associated with higher than normal uric acid levels. This syndrome is characterized by the deficiency of hypoxanthine-guanine phosphoribosyltransferase and is manifested clinically by mental retardation, abnormal muscle movements, and behavioral problems such as self-mutilation and aggressiveness.
Many other analytes are also assayed to monitor the development and treatment of many clinical conditions. However, the assay of such analytes in complex biological fluids is difficult. In many cases, it is difficult to identify many of the substances present in these fluids, even with a sensitive separation technique such as capillary electrophoresis. For example, separation of urine by capillary electrophoresis results in more than fifty readily recognizable peaks, and only a minority of these peaks have so far been identified. Because of the presence of components in urine such as proteins and salts, the migration time of each species present can be affected drastically when compared to an electropherogram of a pure sample of the substance.
The difficulty of identifying such analytes is even greater in serum, because the protein concentration in serum is approximately three orders of magnitude greater than it is in urine, which causes a substantially greater degree of interference and masking.
Therefore, there is a need for an analytical method that detects analytes in complex biological fluids such as serum and urine, and that overcomes the interference introduced by salts and proteins. Such a method should be able to identify and quantitate a wide variety of analytes and do so specifically, avoiding cross reactions. The method should also be able to distinguish between compounds of similar structure such as the amino acids aspartate and glutamate, or steroids with the same ring structure and different substituents. Furthermore, the method should be easy to carry out, give rapid results, and use relatively small samples compatible with current clinical practice.